This project focuses on two serine threonine protein kinases, GCK and GCKR and a tyrosine kinase PYK2. GCK and GCKR belong to a subfamily of protein kinases, which also includes GLK, HPK1, and NIK. They are characterized by an N-terminal kinase domain related to the yeast STE20 protein kinase, a large C-terminal regulatory domain, and the ability to activate the stress- activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. This pathway is activated by many cellular stresses, the inflammatory cytokine tumor necrosis factor (TNF) and CD40 ligand (CD40L). CD40L is present on activated T lymphocytes and interacts with its counter-receptor CD40, which is expressed by many of the important cells in the immune system, including dendritic cells, monocytes, and B- lymphocytes. Previously, we showed that GCK and GCKR are major intermediaries in CD40 and TNF-mediated SAPK activation, and that signaling through these receptors leads to the formation of a trimolecular complex between GCKR, TRAF2, and TANK. CD40 and TNFa-induced activation of germinal center kinases and the subsequent activation of stress activated protein kinases requires TRAF2. While the TRAF2 TRAF domain binds GCK and GCKR, the RING finger domain is needed for their activation. We have found that GCKR activates the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNFa- and TRAF2-mediated GCKR and SAPK activation. TNFa signaling leads to TRAF2 polyubiquitination, TRAF2 oligomerization, and the recruitment, ubiquitination, and activation of GCKR, all sensitive to disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine-63 linked polyubiquitin chain by Ubc13/Uev1A is required for TNFa-mediated GCKR and SAPK activation. A screen for molecules that bind GCKR has resulted in the identification of a panel of strong interacting proteins that includes a number of adaptor proteins such as Crk and CrkL as well as several proteins that participate in the Ras- signaling pathway. To better understand the role of GCK and GCKR in vivo, the murine GCK and GCKR gene have been isolated. Both genes are complicated spanning nearly 100 kilobases with more than 30 exons. This information has been used to create gene targeting constructs. The ES cells in which one allele of the GCKR gene has been inactivated have been created and are being using used to develop mice that are GCKR deficient. Both GCK and GCKR -/- mice have been created. Neither mutation dramatically alters mouse development as GCK and GCKR deficient mice are born with a normal Mendelian frequency. Studies are in progress to characterize the effects of loss of GCK or GCKR on immune cell function, hematopoiesis, and signaling through TNF related receptors. In addition studies with mice in which both GCKR and GCK have been targeted are planned. The tyrosine kinase PYK2 links signals generated by G-protein coupled receptors to downstream signaling pathways. One type of G-protein, G13alpha, potently activates PYK2 kinase activity, which leads to the activation of a small GTPase called RhoA, which in turn stimulates morphological changes in cells and triggers a conserved signaling pathway important for cell growth. The activation of G-proteins in the cells leads to a transient association between PYK2 and G13alpha. A pathway that leads from Gqalpha to the activation of NF-kappaB has been found to depend upon activation of PYK2 as well as the lipid kinase PI-3 kinase and the serine threonine kinase Akt.